Worm CRISPR
This is a work in progress. For a published protocol, see Arribere et al. 2014 PDF. Also see the new protocols from Paix et al. 2016 PDF (PCR template info in Paix et al. 2014 PDF and protocols, and Cas9 Protein info in Paix et al. 2015 PDF and protocols). Design guide RNAs. Can use a website, but really only need to find a GG as close as possible to where you want to edit. GGNGG probably works much better (Farboud et al. 2015 PDF). You will need the 19 nucleotides preceding NGG, and also the sticky ends to facilitate cloning. Order a pair of IDT oligos to use for cloning into DR274 MN3055 plasmid following the example below: :Site in MN’s DR274 U6 3055 :5’ ATGTTTGagagaccaaggatccgggtctcaGTTTTAG 3’ :3’ TACAAACtctctggttcctaggcccagagtCAAAATC 5’ :Guide #3 is on positive strand (cuts 10nt away from desired spot) :Genome: CCAGTCTAAGCAATAAAAT TGG :Forward Oligo: 5’ TTT G CCAGTCTAAGCAATAAAAT 3’ :Reverse Oligo: 3’ GGTCAGATTCGTTATTTTA CAAA 5’ : (5’ AAAC ATTTTATTGCTTAGACTGG 3’) :Order from IDT as: :Guide3F TTTGCCAGTCTAAGCAATAAAAT 25nm STD :Guide3R AAACATTTTATTGCTTAGACTGG 25nm STD Once the oligos come in, dilute them to 100uM in the tube, then dilute 1:10 into another tube to get 10uM. Anneal the two together at a final concentration of 2uM (4ul Fwd, 4ul Rev, 12ul water) on the anneal cycle “crispr primer anneal faster” or any way to slowly cool from 95C to 37C. Digest the backbone (empty DR274 MN3055) with BsaI-HF. Make sure it is as complete as possible (e.g. overnight at 37C) :Water 41ul :Plasmid (~1ug) 3ul (of 334ng/ul miniprep) :CutSmart 10x buffer 5ul :BsaI-Hf 1ul Purify the backbone, either by running on a 0.8% Low-Melt agarose gel (Sea-Plaq) and gel purifying, or, According to MN, running it over a DNA column is enough to lose the tiny insert. So as long as the digest is complete enough, this should work. Mine was at 3.4ng/ul. Ligate '''the annealed oligos and the purified backbone, incubate at 15C for ~1hr. Also perform a control with only the backbone (no insert). :water 13.5ul :backbone 6ul (at 3.4ng/ul) :10x NEB ligation buffer 2.5ul :annealed insert 2ul :NEB T4 DNA ligase 1ul '''Transform into DH5alpha: :10-100ul competent cells :5ul ligation mix ::vortex briefly ::incubate 20-40 minutes on ice ::heat shock 40 seconds at 42C (on Nick’s bench) ::incubate on ice 10 minutes :Add 1ml SOC ::incubate 1hr at 37C with rotation or light shaking : plate 100ul on LB+Kan at 37C and keep the remainder at 4C Pick colonies and screen with colony PCR + BamHI digest (OPTIONAL) :PCR 1rxn __rxns :water 8.4ul :10x Taq. buffer 1ul :10mM dNTPs 0.2ul :10uM primer M13F 0.2ul 5’-TGTAAAACGACGGCCAGT -3’ :10uM primer Rev 0.2ul 5’-CATTCACCAAAGCTCTATCCGAAC -3’ :Template colony :Taq 0.05ul :: aliquot 10ul/well, add a colony to each well :Cycler :denature 95C 5min :denature 95C 30sec <---- :anneal 58C 60sec | 35x :extend 68C 60sec ---- :extend 68C 5min :hold 12C hold :Digest 1rxn __rxns :water 4.3ul :10x cut smart buffer 0.5ul :BamHI-HF 0.2ul :PCR product 10ul ::Aliquot 5ul ::Incubate at 37C ::Run out on 2% agarose gel, look for loss of cut site Grow up guideRNA plasmid colonies overnight at 37C in 5ml LB with shaking Miniprep '''from 3ml (expect 200-400ng/ul) :Double digest with BamHI and EcoNI :Digest 1rxn __rxns :water 8.6ul :miniprep DNA 5ul :10x Cut Smart buffer 1ul :BamHI-HF 0.2ul :EcoNI 0.2ul ::aliquot 10ul ::Incubate at 37C (4 hours was sufficient) ::Run out on 0.8% agarose gel Sequence 2 different minipreps of each guide, just to be sure :0.8ul miniprep (PNACL requests 500-1000ng per kb of plasmid) :3.2ul 1uM primer ZK30 (PNACL requests 3.2pmol of primer) :: 5’-CATTCACCAAAGCTCTATCCGAAC -3’ : 8ul water (PNACL requests 12ul total volume) *Also grow up and '''miniprep Cas9 plasmid MN3143 and dpy-10 gRNA plasmid or 3102 *Obtain ssDNA oligo to target location of dpy-10(cn64) lesion (homologous recombination (HR) template) *Design and obtain (from IDT) ssDNA oligo to target your favorite gene (HR template) *Make sure your oligo changes the PAM or otherwise prevents Cas9 re-cutting Inject (here is an example 20ul recipe) 50 ng/ul Cas9 MN3075 193.3ng/ul -- 5.17ul 20ng/ul dpy-10 sgRNA MN3102 223.6ng/ul -- 1.79ul 500nM dpy-10 repair oligo -- 0.4ul 40ng/ul yfg-1 guide 1 252ng/ul -- 3.17ul 40ng/ul yfg-1 guide 2 326ng/ul -- 2.45ul 600nM yfg-1 repair oligo 25uM -- 0.48ul ddH2O -- 6.54ul Note: yfg-1 is Your Favorite Gene Pick Rol F1, use PCR or phenotype to identify hets Pick non-Rol F2, use PCR or phenotype to identify homozygous edited worms Sequence homozygous F2s